While it is clear that children and adults living in endemic areas do develop resistance to this disease, albeit slowly, the mechanisms responsible for this are not well understood. In 2009 the Long laboratory built upon the foundation established in the previous year to explore the interface between the malaria parasite and the hosts immune system. To accomplish this we have employed both clinical research studies in Mali and several different rodent models of malaria infection. To develop the clinical research, we have collaborated with Dr. Rick Fairhurst of LMVR and Dr. Mahamadou Diakite of the MRTC in Mali to continue a longitudinal study of 1300 children of various ages in 3 villages in Mali. This study was initiated in 2008 and will continue for 5 years so that we can construct a comprehensive picture of the development of adaptive immune responses in children in this malarious area of Mali. A subset of these children is being followed for detailed T cell responses and development of humoral immune responses. In addition, all children have been typed for a series of hemoglobin and red cell polymorphisms by the Fairhurst laboratory;even from the first year of analysis it is already apparent that children with HbS genotype are protected against malaria in this population. We believe that these genetic variants provide a unique window into the acquisition of anti-malarial immunity and are pursuing the hypothesis that comparison of the immune responses of HbS children with other children lacking this genotype will allow us to identify factors that make a major contribution to this protection. We have tested paired plasma samples from the 2008 subset of Malian children against a series of blood stage parasite proteins and these results are being analyzed and compared with those from a larger cohort of children selected in 2009. Similarly we have determined antibody titers to a range of parasite antigens in adults from 3 different ethnic groups - the Malinke, Dogon and Fulani. As a counterpart we are also studying the functional activity of these antibodies we have also tested the activity of purified IgG from these populations in a standardized in vitro parasite growth inhibition assay. We have also continued to refine the analysis of different human CD4+ T cell subsets including T memory cells and multifunctional T cells. We took advantage of the availability of cells from naive vaccine volunteers in the US to develop analytic tools such as intracellular cytokine staining of T cells specific for various blood-stage parasite antigens. We have completed analysis of a clinical trial of the blood-stage antigen PfAMA1 in naive volunteers employing Alhydrogel with CPG to assess the impact of addition of a TLR9 agonist to this formulation. We then extended our analysis of malaria-specific memory T cells to adults and children in Mali, identifying AMA1 specific memory cells in adults in Mali. We are also continuing some studies in mice using rodent malaria parasites to examine the role of multifunctional CD4+ T cells in protective responses after immunization with specific malaria proteins. We are also testing some novel blood stage vaccine candidates in these models and testing various immunologic effector functions. We have also made significant progress in 2 new areas for the laboratory. The first is the immunologic analysis of antibody responses to the DBL3X domain of the VAR2CSA protein. This PfEMP1 domain, whose structure was determined by Dr. Kavita Singh, has been implicated in pregnancy associated malaria through binding to chondroitin sulfate A in the placenta. In collaboration with Dr. Singh we have produced monoclonal and polyclonal antibodies to this domain and showed that both can recognize the surface of CSA-selected parasitized red cells. We have also shown that antibodies can inhibit the binding of parasitized erythrocytes to CSA. The second new project involves the application of aptamers to search for conserved antigenic determinants on the surface of malaria infected erythrocytes. We have prepared complex DNA aptamer libraries and conducted a series of selections on various targets. The selected preparations are now being sequenced using next generation technology and we are confirming reactivity of different aptamer families on red cell surfaces.